Improved Cortical Spheroids and Methods of Making the Same

ABSTRACT

Improved hybrid neurovascular spheroids and methods for making the same are provided. In some embodiments of a method for making a hybrid neurovascular spheroid, the method includes i) propagating cortical cells to form a cortical spheroid; ii) propagating endothelial cells to form an endothelial spheroid; iii) propagating mesenchymal stem cells to form a mesenchymal cell culture; and iv) combining the cortical spheroid, endothelial spheroid, and mesenchymal spheroid under conditions to form the hybrid neurovascular spheroid.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.16/821,395, filed Mar. 17, 2020, which claims priority to U.S.Provisional Application No. 62/821,807 filed Mar. 21, 2019. The contentof these applications is incorporated by reference herein.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under Grant No.R03EB020770 awarded by the National Institutes of Health. The governmenthas certain rights in the invention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in XML format and is hereby incorporated byreference in its entirety. Said XML copy, created on Jun. 28, 2023, isnamed 19585_0545_Sequence_Listing_ST_26_XML_Format and is 26000 bytes insize.

BACKGROUND

Brain organoids derived from human induced pluripotent stem cells(hiPSCs) have emerged as powerful model systems for neurological diseasemodeling, drug screening, and for studying Zika virus infections [1-5],which affect over one billion people globally [6], as well asmicrocephaly [3], and lissencephaly [63]. However, the lack ofinteractions with other cell types such as endothelial cells in currentbrain organoids model limits their applications [5, 9]. As neurologicaldiseases, such as blood-brain barrier (BBB) breakdown and dysfunction inAlzheimer's and stroke, involve multiple cell types, in vitro modelssuch as brain organoids must include relevant cell types to reconstructcellular microenvironment [13]. As the vascular system is an essentialcomponent of brain tissue, incorporating neural-vascular interactions inforebrain organoids is an important step in developing brain organoidsin vitro. To date, the actual vascularization was only achieved in vivo[18, 19, 84]. In vitro vascularization needs accurate design of ECMamounts and the structure (insoluble) and the soluble secreted factors.Whole brain organoids derived from hiPSCs in Matrigel with isogenic ECsor coated day 34 spheroids with ECs have been used to achieve in vitroand in vivo vascularization [85]. In another study from Mansour et al.(2018), vascularized brain organoids in vivo show the integration ofmicroglia and functional neuronal networks and blood vessels [84]. Allthese studies indicate that neural-vascular interactions areindispensable for modeling neurological diseases and screening drugsthat require 3-D brain tissue structure [64]. This is particularly truefor the use of diseased hiPSC lines such as in Alzheimer's disease [31].Alzheimer's-patient derived cortical organoids containing vascular cellswould be important to recapitulate a neurodegenerative microenvironmentand investigate its response to potential drug treatments.

However, generating brain-region specific organoids with definedstructure and function remains a critical challenge because theheterotypic cell-cell interactions to mimic the human brain have not yetbeen fully understood [7-9]. Recently, fusion of human forebrainspheroids of different regions (e.g., human dorsal spheroids withventral spheroids) has been investigated to model interneuron migrationand the interactions of different neuronal subtypes [10-12]. However,the interactions of neuronal cells with other cell types, such asendothelial cells, have not been fully studied in brain organoids [5].

Cerebral organoids derived from induced pluripotent stem cells (iPSCs)provide novel tools for recapitulating the cytoarchitecture of the humanbrain and for studying biological mechanisms of neurological disorders.However, these cerebral organoids typically do not allow for theobservation of heterotypic interactions of neurovascular units, composedof neurons, pericytes, astrocytes, and brain microvascular endothelialcells, in brain-like tissues.

Accordingly, improved cerebral organoids and methods of making the sameare needed.

BRIEF SUMMARY

Improved hybrid neurovascular spheroids and methods for making the sameare provided. In one aspect, a method is provided for making a hybridneurovascular spheroid, wherein the method includes i) propagatingcortical cells to form a cortical spheroid; ii) propagating endothelialcells to form an endothelial spheroid; iii) propagating mesenchymal stemcells to form a mesenchymal cell culture; and iv) combining the corticalspheroid, endothelial spheroid, and mesenchymal spheroid underconditions to form the hybrid neurovascular spheroid.

In some embodiments, at least a portion of the mesenchymal cell cultureis combined with the cortical spheroid first, to form a mesenchymalcell-cortical spheroid which is then combined with the endothelialspheroid to form the hybrid neurovascular spheroid. In some otherembodiments, at least a portion of the mesenchymal cell culture iscombined with the endothelial spheroid first, to form a mesenchymalcell-endothelial spheroid which is then combined with the corticalspheroid to form the hybrid neurovascular spheroid.

In some embodiments, the cortical cells are human iPSC-derived corticalneural progenitor cells (iNPC), cortical cells are pericytes,astrocytes, or microglia. In some embodiments, the endothelial cells arehuman endothelial cells (iEC) and/or the mesenchymal stem cells arehuman mensenchymal stem cells (hMSC).

In some embodiments, the one or more of the cortical cells, endothelialcells, and mesenchymal stem cells are propagated in a hydrogelcomprising Geltrex, hyaluronic acid (HA), or any combination thereof. Insome embodiments, the method further includes treating one or more ofthe spheroids with a CXCR4 antagonist, including AMD3100.

In another aspect, a hybrid neurovascular spheroid is provided thatincludes cortical cells, endothelial cells, and mesenchymal stem cells.In some embodiments, the hybrid neurovascular spheroid displaysupregulated secretion levels of one or more cytokines VEGF-A, PGE2, andTBF-01 relative to a comparative hybrid neurovascular spheroid preparedby directly mixing cultures of cortical cells, endothelial cells, andmesenchymal stem cells.

In some embodiments, the hybrid neurovascular spheroid displaysincreased expression of one or more of TBR1, Nkx2.1, matrix remodeling,MMP2, MMP3, Notch-1, GLUT-1, CD31, or ZO-1 genes relative to acomparative hybrid neurovascular spheroid prepared by directly mixingcultures of cortical cells, endothelial cells, and mesenchymal stemcells.

In some embodiments, the hybrid neurovascular spheroid has a ratio basedon initial seeded cell numbers of the cortical cells to the endothelialcells to the mesenchymal stem cells wherein the ratio is from about4:2:0 to about 1:2:3.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1D depict the endothelial differentiation from hiPSCs insuspension to generate iEC spheroids.

FIGS. 2A-2C depict the characterization of neural differentiation fromhiPSCs in suspension.

FIG. 3 depicts methods to form hybrid spheroids according to embodimentsof the present disclosure.

FIG. 4 depicts phase contrast images of iNPC-iEC-MSC spheroidsmorphology at day 1, 3, 5, and 7 (total day 15-21).

FIGS. 5A-5B depict overlay of phase contrast images (iNPCs and iECs)with fluorescent images (hMSCs) of hybrid spheroids.

FIGS. 6A-6C depict a schematic illustration of calculation of squaredaspect ratio of contact length between two aggregates over maximumdiameter and aggregation kinetics.

FIGS. 7A-7B depict the aggregation kinetics analyzed in (Ei) and (Eii),respectively.

FIGS. 8A-8B depict overlay of phase contrast images (iNPC spheroids andiEC spheroids) with fluorescent images (hMSCs) of iNPC-iEC-MSC spheroidswith Geltrex at 5% or 10% and aggregation kinetics.

FIGS. 9A-9B depict overlay of phase contrast images with fluorescentimages of iNPC-iEC-MSC spheroids with hyaluronic acid (HA, 0.025 wt % or0.05 wt %) and aggregation kinetics.

FIGS. 10A-10B depict overlay of phase contrast images and fluorescentimages of iNPC-iEC-MSC spheroids with the treatment of ROCKi Y27632 (20μM or 40 μM); and aggregation kinetics of hybrid spheroids.

FIGS. 11A-11D depict the metabolic activity and DNA content of hybridspheroids in suspension.

FIG. 12 depicts DNA content of hybrid spheroids with the treatment of0.025 wt % hyaluronic acid (HA).

FIGS. 13A-13D depict cytokine secretion by hybrid spheroids duringneural differentiation from culture supernatants collected and measuredby enzyme-linked immunosorbent assay (ELISA) for different spheroids atday 21.

FIG. 14 depicts VEGF-A secretion of an iEC control.

FIG. 15 depicts morphology and histology characterizations for early(day 21) and late (day 47) stage hybrid spheroids.

FIGS. 16A-16D depict electrophysiological properties of hybridspheroids.

FIGS. 17A-17C depict RT-PCR analysis for iEC and hMSC controls comparedto iNPC group.

FIGS. 18A-18B depict investigation of neurogenesis of hybrid iNPC-MSCspheroids on 2D surface with iEC layer.

FIG. 19 depicts the effects of AMD3100 treatment on MTT activity ofhybrid spheroids.

FIG. 20 depicts a schematic illustration of a process of making hybridcortical spheroids according to an embodiment of the present disclosure.

FIGS. 21A-21D depict histology images of early and late stage hybridspheroids and quantification by flow cytometry.

FIGS. 22A-22I depict the results of RT-PCR analysis of gene expressionof hybrid spheroids.

FIGS. 23A-23D depict the effects of AMD3100 treatment on the aggregationkinetics of hybrid spheroids.

DETAILED DESCRIPTION

Hybrid neurovascular spheroids are provided that includes corticalcells, endothelial cells, and mesenchymal stem cells. The spheroid, or3-D cell culture, is a fusion of aggregates of each cell type. That is,an aggregate of cortical cells, and an aggregate of endothelial cells,and an aggregate of mesenchymal stem cells, wherein the aggregates arefused into direct contact in a 3-D architecture. This spheroid structureis distinct from a 3-D culture in which the different cell types aremixed together before forming the 3-D architecture.

Neural-vascular interactions, known as neural-vascular units (NVU), playan important role in brain structure and function [13]. It is believedthat organ-specific endothelial cells secrete a unique set of growthfactors that regulate tissue morphogenesis into desired tissue types[14]. Vascular cells can form spheroids to assemble blood vessels or asbuilding blocks for scaffold-free tissue fabrication [15, 16]. In vitrovascularization of organoids has been attempted for cardiac organoids,showing enhanced cardiac cell function [17]. In vivo vascularization oforganoids was realized for iPSC-derived organ buds, in which the mixediPSC-derived progenitors and endothelial cells efficientlyself-organized into functional and vascularized liver or kidney in vivorespectively [18, 19]. In particular, the blood-brain barrier (BBB) isinvolved in various neurological diseases development, drugadministration and nutrient transport [13, 20]. Functional BBB modelsrequire the interaction of brain microvascular endothelial cells (ECs),astrocytes, neurons, and pericytes, which can be realized usinghiPSC-derived cells [21-24].

Mesenchymal stem cell (MSC)-driven condensation has been observed inorgan buds formation based on iPSC-derived cells for multiple tissuetypes including kidney, intestine, brain, and heart etc., in thepresence of MSCs [19]. Although it remains unclear if MSC-drivencondensation is due to adhesion molecules expression or cytoskeletonreorganization, the MSCs support organoid formation from multipleaspects. MSCs reside in virtually all adult tissues including brain andthe vicinity of the capillaries, and at least at a subset of MSCs(CD146+CD34⁻) can function as pericytes that are closely associated withvasculature [25-27]. In the context of brain organoids, MSC secretomeare a potent source of trophic factors that are modulators of neurogenicniche and promote angiogenesis and neural differentiation throughtrophic effects (e.g., fibroblast growth factor (FGF)-2, vascularendothelial growth factor (VEGF), brain-derived neurotrophic factoretc.). MSCs also secrete anti-apoptotic and anti-inflammatory factors,e.g., Prostaglandin E2 (PGE2), and ECM proteins [28]. MSCs displayedhigher homing ability to injury sites for neural protection, due to theincreased expression of CXCR-4 [29]. Thus, without intending to be boundby any particular theory, it is believed that the incorporation of ECsand MSCs enables the formation of a pro-neurogenic niche that promotesangiogenesis, neo-brain tissue patterning, and maturation.

Previous studies have assembled hiPSC-derived neural progenitor cells(iNPCs) and human bone marrow MSCs in spheroid culture, showing thatMSCs promote dorsal cortical spheroid formation [30]. The derivation ofcortical spheroids or organoids was also achieved in a suspensionbioreactor and from Alzheimer's patient specific hiPSCs [31-33].

However, without intending to be bound by any particular theory, it isbelieved that vascularization of cortical organoids in vitro throughtri-culture of iNPCs, hiPSC-derived ECs, and human MSCs, may allow forthe investigation of heterotypic neural-vascular-mesenchymalinteractions.

Most previous methods directly mixed endothelial cells with other celltypes for 3-D co-culture systems [17, 19, 64]. Advantageously, unlikedirect mixing methods, the spheroid fusion methods described herein (1)avoid cell dissociation and re-association processes, which could losemany cells; and (2) the hybrid spheroid structure can be pre-controlledwith special compartment arrangements.

The spheroid fusion method has been used for assembly of dorsal andventral forebrain spheroids to study interneuron migration and theassembly of iPSC-derived endothelial progenitor spheroids and smoothmuscle progenitor spheroids [10, 16]. Fusion of spheroids of differentcell types is most likely driven by minimization of interfacial freeenergy and cellular thermodynamics, differential cellular adhesions(e.g., E-cadherin expression), or cortical tension redistribution [28,65, 66]. Without intending to be bound by any particular theory, it isbelieved that the fusion kinetics is affected by ROCK inhibitor, ECMs inthe medium, and the mixing sequence. Without intending to be bound byany particular theory, it is believed that the addition of highconcentration of ROCKi Y-27632 during the initial aggregation delayedthe fusion process, indicating that cortical tension regulates spheroidfusion process, and that actomyosin may play a key role in spheroidfusion [47, 67]. Without intending to be bound by any particular theory,it is believed that the presence of GELTREX™, a soluble form of basementmembrane, (Life Technologies Corporation) (hereinafter “Geltrex”) andHA, an ECM component in the brain, at an appropriate concentrationpromotes spheroid fusion.

Without intending to be bound by any particular theory, it is believedthat, since MSCs condense into the center of the hybrid spheroids,adding MSCs to iNPC spheroids before iEC spheroid fusion (iNPC-MSC-iEC)would constrict MSCs as the iNPC spheroid core. Accordingly, in someembodiments, the present disclosure includes iNPC spheroid and iECspheroid fusion in the presence of MSCs (iNPC-iEC-MSC).

In some embodiments, assembly of vascular spheroids and corticalspheroids as disclosed herein may enhance the glucose transporter,GLUT-1 (specifically expressed in endothelial cells in brain [13]), anda polarized efflux transporter BCBP. In some embodiments, the tightjunction protein ZO-1 may be promoted in the tri-culture, indicatingthat neurovascular co-patterning promotes the specification of iECstoward brain microvascular cells.

Human iPSC-derived 2-D co-culture system of multiple cell types (i.e.,neurons, astrocytes, pericytes and brain microvascular endothelialcells) has been recently used to mimic BBB function with highertrans-endothelial electrical resistance (TEER) properties and study drugpermeability in vitro [21-24]. Neural-vascular interactions result inthe special structure and function of BBB. However, the 3-D BBB modelshave not been well established due to the complex BBB features and thedifficulty of forming micro-vessel structures in 3-D. Some studies usehollow fiber systems with perfusion culture or artificially createmicrochannels for 3-D vascularization [20]. Although this system is notyet an accurate and perfusable 3-D BBB model, the system recapitulatesthe anatomical features of the BBB using human stem cells. The inclusionand characterization of additional cell types (e.g., astrocytes), morecomplex 3-D capillary network (novel biomaterials design), and perfusionflow (use bioreactors or microfluidics) may be explored in the future[68].

Without intending to be bound by any particular theory, it is believedthat neural-vascular interactions may also impact brain tissuepatterning. In some embodiments, hybrid spheroids may exhibit elevatedβ-tubulin III and CD31 expression, as well as higher TBR1 and Nkx2.1gene expression in tri-culture (in particular the 1:2:3 group). Inaddition, it is believed that the “inside-out” development of thecortical superficial and deep layers in the forebrain is faster for thetri-(1:2:3) group compared to other groups. It has been reported thatthe neural differentiation of hPSCs requires the direct association withvascular cells [69] as well as hMSCs, possibly through MAPK and PI3K-Actsignaling (involved FGF-2) [70]. In some embodiments, the tri-culturesystem described herein may promote the expression of Notch-1, the keyprotein in Notch signaling which is responsible for cell-cell contactinteractions involved ECs [71, 72]. In addition, activation of Notchsignaling can promote the neural stem cell self-renewal, glial celldifferentiation, and neuron regeneration [73, 74]. Without intending tobe bound by any particular theory, it is believed that the hybridspheroids described herein may exhibit direct contact among iNPCs, iECs,and hMSCs at a given ratio, which may accelerate development of 3-Dcortical tissue structure containing vascular cells to model human braindevelopment.

The interactions of hMSCs and iNPC have been discussed in [30]. Withoutintending to be bound by any particular theory, it is believed that thepresence of MSCs in addition to iECs was found to enrich ECMlocalization and affect matrix remodeling. As brain ECM has littlefibril ECMs such as collagens [20], the expression of collagen IV inhybrid spheroids was mainly attributed to MSCs. Without intending to bebound by any particular theory, it is believed that the elevated MMP-2and MMP-3 expression indicates the active matrix remodeling, which isrequired for maintaining neural stem cells [62]. It has been found thatthe formation of hMSC aggregates upregulates several types of MMPs(MMP-2, -9, and -1/13) [75], which were reported to enhance neuronaldifferentiation through NF-κB signaling [76]. Without intending to bebound by any particular theory, it is believed that ECM remolding mayalso be an important contributing factor involved in the migration andinvasion of the cells. In some embodiments, the hybrid spheroid, theimmobilized hMSCs after CXCR4 (receptor for CXCL12/stromal-derivedfactor-1 chemokine) inhibition reveals the migration ability of hMSCswithin the fused spheroids.

Secretion of cytokines and neurotrophin is a critical function of hMSCs,which can enhance neurogenesis of hiPSCs [77]. The influence of TGF-β1and PGE2 secreted by MSCs on hiPSC-neural differentiation is known [30].The upregulated TGF-β1 and PGE2 secretion by MSCs promotes Nestin andβ-tubulin III expression. Without intending to be bound by anyparticular theory, it is believed that the main source of VEGF-A ishMSCs, although vascular cells also contribute to VEGF-A secretion.Previous studies have suggested that brain vascular ECs promote neuralcell functionality, such as synaptic activities, via the modulation ofVEGF signaling and that the VEGF receptors were activated by neuralcell-secreted nitric oxide [78, 79]. Without intending to be bound byany particular theory, it is believed that iECs may be a minor source ofVEGF-A (as shown in Bi-(4:2) group) in hybrid spheroids of the presentdisclosure as they are not mature enough compared to human bone marrowMSCs. The elevated VEGF-A in tri-culture (in particular the 1:2:3 group)regulated by Notch signaling is a result of close cell-cell contacts ofneural-vascular-mesenchymal cells through autocrine, paracrine, andjuxtacrine interactions [80, 81].

Without intending to be bound by any particular theory, it is believedthat the cellular ratio indicates that tri-(1:2:3) better promotesneural-vascular interactions than the other groups, indicated by highercytokine secretion, neural patterning marker expression, BBB-relatedgene expression, and the cortical layer separation. The braincomposition showed a 1:3 neuron-to-astrocyte ratio [22], indicating theimportance of accessory cells on neural function. In some embodiments,endothelial cell apoptosis may exist due to upregulated caspase3/7expression and altered mitochondria bioenergetics on 3-D MSC aggregationdue to compaction [82]. An alternative is the use of iPSC-derived MSC(iMSCs) in the tri-culture system as reported by Gao et al. [83].

In some embodiments of the present disclosure, hiPSC-derived vascularspheroids and hiPSC-derived neural cortical spheroids are assembled inthe presence of hMSCs to study neurovascular interactions. Withoutintending to be bound by any particular theory, it is believed that thepresence of hMSCs promotes cortical neural differentiation, layerseparation, cytokine secretion, and cell-cell communication. Withoutintending to be bound by any particular theory, it is believed that thepresence of iECs provides the BBB-related properties inside the corticalspheroids or organoids. In some embodiments, hybrid spheroids mayexhibit elevated Notch signaling, matrix remodeling proteins, andsecretion of VEGF-A. Without intending to be bound by any particulartheory, it is believed that this may contribute to the acceleratedcortical tissue development.

In some embodiments of a method for making a hybrid neurovascularspheroid, the method includes i) propagating cortical cells to form acortical spheroid; ii) propagating endothelial cells to form anendothelial spheroid; iii) propagating mesenchymal stem cells to form amesenchymal cell culture; and iv) combining the cortical spheroid,endothelial spheroid, and mesenchymal spheroid under conditions to formthe hybrid neurovascular spheroid.

In some embodiments, the step of combining the cortical spheroid,endothelial spheroid, and mesenchymal spheroid under conditions to forma fused spheroid includes i) combining at least a portion of themesenchymal cell culture with the cortical spheroid to form amesenchymal cell-cortical spheroid; and ii) combining the mesenchymalcell-cortical spheroid with the endothelial spheroid to form the hybridneurovascular spheroid.

In some embodiments, the step of combining the cortical spheroid,endothelial spheroid, and mesenchymal spheroid under conditions to forma fused spheroid includes i) combining at least a portion of themesenchymal cell culture with the endothelial spheroid to form amesenchymal cell-endothelial spheroid; and ii) combining the mesenchymalcell-endothelial spheroid with the cortical spheroid to form the hybridneurovascular spheroid.

In some embodiments, the cortical cells are human iPSC-derived corticalneural progenitor cells (iNPC). In some embodiments, the cortical cellsare pericytes, astrocytes, or microglia.

In some embodiments, the endothelial cells are human endothelial cells(iEC). In some embodiments, the mesenchymal stem cells are humanmensenchymal stem cells (hMSC).

In some embodiments, the ratio based on initial seeded cell numbers ofthe cortical cells to the endothelial cells to the mesenchymal stemcells is from about 4:2:0 to about 1:2:3.

In some embodiments, the cortical cells, endothelial cells, andmesenchymal stem cells are propagated in separate low-attachment wellplates or in other suitable vessels.

In some embodiments, the one or more of the cortical cells, endothelialcells, and mesenchymal stem cells are propagated in a hydrogelcomprising Geltrex, hyaluronic acid (HA), or any combination thereof. Insome embodiments, the one or more cortical cells, endothelial cells, andmesenchymal stem cells are propagated in a hydrogel comprising at leastabout 5 wt. % Geltrex and 0.025 wt. % HA.

In some embodiments, the method further includes treating one or more ofthe spheroids with a CXCR4 antagonist. In some embodiments, the CXCR4antagonist is AMD3100.

In some embodiments, a hybrid neurovascular spheroid is provided thatincludes cortical cells, endothelial cells, and mesenchymal stem cells.

In some embodiments, the hybrid neurovascular spheroid displaysupregulated secretion levels of one or more cytokines VEGF-A, PGE2, andTBF-01 relative to a comparative hybrid neurovascular spheroid preparedby directly mixing cultures of cortical cells, endothelial cells, andmesenchymal stem cells.

In some embodiments, the hybrid neurovascular spheroid displaysincreased expression of one or more of TBR1, Nkx2.1, matrix remodeling,MMP2, MMP3, Notch-1, GLUT-1, CD31, or ZO-1 genes relative to acomparative hybrid neurovascular spheroid prepared by directly mixingcultures of cortical cells, endothelial cells, and mesenchymal stemcells.

In some embodiments, the cortical cells are human iPSC-derived corticalneural progenitor cells. In some embodiments, the endothelial cells arehuman endothelial cells. In some embodiments, the mesenchymal stem cellsare human mensenchymal stem cells (hMSC).

In some embodiments, the hybrid neurovascular spheroid has a ratio basedon initial seeded cell numbers of the cortical cells to the endothelialcells to the mesenchymal stem cells wherein the ratio is from about4:2:0 to about 1:2:3.

In some embodiments, the hybrid neurovascular spheroid includes one ormore of cortical cells, endothelial cells, and/or mesenchymal stem cellsthat are propagated in a hydrogel. The hydrogel may include Geltrex,hyaluronic acid, or a combination thereof.

The hybrid neurovascular spheroids made by the methods described hereinhave a variety of applications. For example, the spheroids may be usedas a research tool, including as a research model for testingpharmaceuticals or other substances' effects on healthy or diseasedneurovascular tissue. Furthermore, the spheroids have severalapplications in the field of tissue engineering, including the formationof tissue for medical treatment.

The spheroids and methods can be further understood with the followingnon-limiting examples.

EXAMPLES

In each of the examples described below, the human iPSK3 cells used werederived from human foreskin fibroblasts transfected with plasmid DNAencoding reprogramming factors OCT4, NANOG, SOX2 and LIN28 (kindlyprovided by Dr. Stephen Duncan, Medical College of Wisconsin, and Dr.David Gilbert, Department of Biological Sciences of Florida StateUniversity) [34, 35]. The human iPSK3 cells were maintained in mTeSRserum-free medium (StemCell Technologies, Inc., Vancouver, Canada) on6-well plates coated with growth factor reduced Geltrex (LifeTechnologies). The cells were passaged by Accutase dissociation every5-6 days and seeded at 1×10⁶ cells per well of 6-well plate in thepresence of 10 μM Y27632 (Sigma) for the first 24 hours [36-38].

Each experiment was carried out at least three times (using differentbatches of cells) with triplicate samples (in some cases spheroids werepooled from more than 12 wells) in each experiment. The representativeexperiments were presented and the results were expressed as[mean±standard deviation]. To assess the statistical significance,one-way ANOVA followed by Fisher's LSD post hoc tests were performed. Ap-value<0.05 was considered statistically significant.

Example 1: hMSC Culture Formation and iEC and iNPC Spheroid Formation

To generate a human MSC (hMSC) culture, standardized frozen hMSCs frommultiple donors were obtained from the Tulane Center for Gene Therapyand cultured as previously described [39, 40]. The hMSCs were isolatedfrom the bone marrow of healthy donors ranging in age from 19 to 49years based on plastic adherence, negative for CD34, CD45, CD117 (allless than 2%) and positive for CD29, CD44, CD49c, CD90, CD105, and CD147markers (all greater than 95%), and possessed tri-lineagedifferentiation potential upon induction in vitro [41, 42]. hMSCs wereexpanded at a density of 1.7×10³ cells/cm² using MEM (Invitrogen) mediumsupplemented with 10% fetal bovine serum (FBS) and 1%penicillin/streptomycin. At approximately 80% confluence, adherent cellswere harvested with 0.25% trypsin-EDTA (Sigma-Aldrich) and furtherpropagated.

To generate an endothelial cell spheroid (iEC), undifferentiated iPSK3cells were seeded in U-bottom ultra-low-attachment (ULA) 96-well plates(Corning Inc.) at 1×10⁴ per well (unless otherwise noted) indifferentiation medium composed of RPMI plus 2% B27 (Life Technologies)for 3 days. Y27632 (10 μM) was added during the seeding and removedafter 24 h. Then Wnt activator CHIR99021 (10 μM, StemCell TechnologiesInc.) was added to the culture medium for 5 days followed by another 6days in medium without CHIR99021 [43-45]. iEC aggregates (day 14) werecharacterized or transferred to the wells containing iNPC spheroids. TheiEC spheroids expressed endothelial markers CD31 and VE-cadherin, asillustrated in FIGS. 1A-1B. Specifically, FIG. 1A is a schematicillustration of endothelial differentiation protocol. FIG. 1B showsvascular markers, CD31 and VE-cadherin (VECAD), expression wasquantified by flow cytometry. Solid line: negative control; dashedlines: marker of interest from two differentiations. Day 11 vascularspheroids were re-plated for one day and immunocytochemistry wasperformed for vascular markers (not shown). Day 14 vascular spheroidswere harvested and prepared for histology sections (not shown).Haemotoxylin and Eosin (H&E) staining and CD31 staining was performed(not shown). Vascular network formation was observed for day 14 iECspheroids re-plated on 1:1 Geltrex-coated surface in endothelial mediumand cultured for 7 days (FIG. 1C). Scale bar: 100 μm. Briefly, 24-wellplates were coated with 200 μL/well 1:1 diluted Geltrex for more than 30min. The cells were plated at 5×10⁵ cells on Geltrex-coated plates in500 μL EGM-2 medium (Lonza, for endothelial cells) and incubated at 37°C. in 5% CO₂ for 7 days. Cell morphology was photographed by aphase-contrast microscope (not shown). VE-cadherin staining of iEC after30 days of differentiation was performed (not shown). VE-cadherin(VECAD), expression in day 17 iECs was quantified by flow cytometry(FIG. 1D).

To generate a cortical iNPC spheroid, undifferentiated iPSK3 cells(0.5-2×10⁴ cells) were seeded into U-bottom ULA 96-well plates in neuraldifferentiation medium composed of DMEM/F-12 plus 2% B27 in the presenceof Y27632 (10 μM). The aggregates were treated with 10 μM SB431542(Sigma) and 100 nM LDN193189 (Sigma) for 7 days [31]. At day 7, thespheroids were treated with retinoic acid (RA) (2 μM, Sigma) and FGF-2(25 ng/mL, Life Technologies) and grown in neural medium for another 7days [37]. For maturation, cortical spheroids were maintained in neuraldifferentiation medium without growth factors for additional 7-38 days.The NPC spheroids expressed various neuronal markers such as Nestin,TBR1 and β-tubulin III, as shown in FIGS. 2A-2B [33]. Specifically, FIG.2A shows phase contrast images of iNPC spheroids morphology over 13days. Scale bar: 200 μm. Confocal images of cortical neuron marker TBR1and hindbrain marker HOXB4 were taken for day 14 iNPC spheroids (notshown). Neural progenitor marker expression of Nestin and TBR1 ofre-plated day 14 iNPC spheroids was observed (not shown). FIG. 2B showsneurite outgrowth and axon extension indicated by β-tubulin IIIexpression and phase contrast images of neurons after replating. Scalebar: 100 μm. Different phenotypic marker expression on day 28 wasobserved using Glutamate/Tau and Synapsin-1 (SYN-1)/β-tubulin III,related to neurogenesis. (not shown). FIG. 2C shows flow cytometryanalysis of Nestin and β-tubulin III (day 20). Solid line: negativecontrol; dashed line: marker of interest.

Next, using these individual iNPC, MSC, and iEC spheroids, hybridspheroids were formed as described in Examples 2 and 3, below. FIG. 3depicts methods to form hybrid spheroids according to embodiments of thepresent disclosure including a schematic illustration of derivingendothelial cell (iEC) spheroids from hiPSCs and a schematicillustration of generating hybrid spheroids from iNPCs. hMSCs were addedto iNPCs for iNPC-MSC-iEC spheroids (method A), before iEC transfer. OrhMSCs were added to the well containing both iNPC spheroids and iECspheroids for iNPC-iEC-MSC spheroids (method B). hMSCs were labeled withCellTracker Red.

Example 2: Hybrid iNPC-MSC-iEC Spheroid Formation

Human MSCs were pre-labeled with CellTracker Red (2.5 μM, LifeTechnologies) for 30 min unless otherwise noted. Different numbers ofhMSCs were added to day 7 iNPC spheroids (with different seedingdensities) for a total of 2×10⁴ cells (FIG. 3 ) or directly mixed at day0 [30]. The ratios of hiPSC: hMSCs were 4:0, 3:1, 2:2, and 1:3 based oninitial seeded cell numbers. Then, the day 14 iEC spheroids weretransferred into the wells containing day 14 iNPC-MSC spheroids.Different cell ratios for generating hybrid spheroids wereiNPC:iEC:MSC=4:2:0, 3:2:1 2:2:2, and 1:2:3 (total 4×10⁴ per well,n=3-12), referred as Bi-(4:2), Tri-(3:2:1), Tri-(2:2:2), and Tri-(1:2:3)respectively. For some experiments, iNPC only [32, 37], iEC only [16,46] or MSC only spheroids [28, 47, 48] were used as controls. The hybridiNPC-MSC-iEC spheroids were maintained in neural differentiation mediumfor additional 7 days for aggregate fusion.

The iNPC-MSC spheroids and iEC spheroids fused into the aggregates withsquared aspect ratio of 0.5-0.7 after 7 days.

Example 3: Hybrid iNPC-iEC-MSC Spheroid Formation

In this example, day 14 iEC spheroids were transferred into the wellscontaining day 14 iNPC spheroids first (FIG. 3 ). Immediately after thetransfer, hMSCs were added into the wells at different cell densities tomaintain ratios of iNPC:iEC:MSC=4:2:0, 3:2:1, 2:2:2, and 1:2:3 (n=3-12).For long-term cultures, day 14 iEC spheroids were transferred into thewells containing day 14 iNPC spheroids to allow aggregate fusion. Then,at 7 days before harvesting, hMSCs were added into the wells atdifferent cell densities to maintain ratios of iNPC:iEC:MSC=4:2:0, 3:2:12:2:2, 1:2:3, and 0:2:4. The total culture length ranged from 21 days to52 days.

For iNPC-iEC-MSC spheroids, hMSCs were added into the well of day 14iNPC-iEC hybrid spheroids and cultured for another 7 days or MSCs andiEC spheroids were added together to the wells containing iNPC spheroids(FIGS. 4, 5A-5B, and 6A-6C).

Specifically, FIG. 4 depicts phase contrast images of iNPC-iEC-MSCspheroids morphology at day 1, 3, 5, and 7 (total day 15-21). iECspheroids and MSCs were added to the preformed iNPC aggregates at thesame time. Scale bar: 400 μm.

FIGS. 5A-5B depict overlay of phase contrast images (iNPCs and iECs)with fluorescent images (hMSCs) of hybrid spheroids with (FIG. 5A) orwithout (FIG. 5B) ROCKi Y27632 (10 μM) when MSCs were added to theculture one week after fusion of iNPC spheroids and iEC spheroids. Scalebar: 400 μm.

FIG. 6A is a schematic illustration of calculation of squared aspectratio of contact length between two aggregates over maximum diameter

$\left( \frac{Lneck}{L\max} \right)^{2}.$

The aggregation kinetics were evaluated by the squared aspect ratio over7 days (FIG. 6B) and the inter-sphere angle formed by the two aggregates(FIG. 6C).

hMSCs were integrated with the iNPC-iEC hybrid spheroids and migratedtoward the spheroid center. The squared aspect ratio was in the range of0.9-1.0 (FIGS. 7A-7B). Specifically, FIGS. 7A-7B depict the aggregationkinetics were analyzed in (Ei) and (Eii), respectively. *indicatesp<0.05 for the different test conditions.

The low concentration of ROCKi did not impact the integration of MSCsinto iNPC-iEC spheroids. Due to the higher squared aspect ratio(indicating good fusion kinetics), iNPC-iEC-MSC spheroids were mainlyused in the following experiments.

The effects of Geltrex (similar to Matrigel, commonly used in organoidformation [3, 54]) or hyaluronic acid (HA) (an ECM component in brain[55]) on the fusion of neural spheroids and vascular spheroids wasinvestigated (FIGS. 8A-8B and 9A-9B).

Specifically, overlay of phase contrast images (iNPC spheroids and iECspheroids) with fluorescent images (hMSCs) of iNPC-iEC-MSC spheroidswith Geltrex at 5% or 10% and aggregation kinetics of hybrid spheroidsembedded in Geltrex (5% or 10%) was depicted (not shown).

Overlay of phase contrast images with fluorescent images of iNPC-iEC-MSCspheroids with hyaluronic acid (HA, 0.025 wt % or 0.05 wt %) andaggregation kinetics of hybrid iNPC-iEC-MSC spheroids embedded in 0.025or 0.05 wt % HA was depicted (not shown).

Quantification of squared aspect ratios over 7 days showed a good fusiontendency for 5% Geltrex (0.7-0.9 by day 7), but not for 10% Geltrex(0.4-0.6 by day 7) (FIGS. 8A-8B). For HA treatment, 0.025 wt % HAresulted in squared aspect ratio in the range of 0.7-0.9. But 0.05 wt %HA treatment resulted in lower squared aspect ratios (˜0.6 by day 7) forTri-(2:2:2) and Tri-(1:2:3) groups (FIGS. 9A-9B).

The tendency of two spheroids to fuse was disrupted by ROCKi Y27632 athigh concentration (FIGS. 10A-10B). Specifically, overlay of phasecontrast images and fluorescent images of iNPC-iEC-MSC spheroids withthe treatment of ROCKi Y27632 (20 μM or 40 μM) was depicted (not shown);and aggregation kinetics of hybrid spheroids were treated with Y27632(20 μM or 40 μM) (FIG. 10A). The time when three cell types wereco-cultured (day 14) was counted as day 0. Day 1-7 equals total day15-21. Scale bar: 400 μm. *indicates p<0.05 for the different testconditions.

The aspect ratios (0.5-0.7) of 20 μM or 40 μM ROCKi treatments showed aplateau over 7 days (FIGS. 10A-10B). Without intending to be bound byany particular theory, it is believed that these results indicate that5% Geltrex and 0.025 wt % HA promote spheroid fusion.

Example 4: Effects of Geltrex, Hyaluronic Acid (HA) Hydrogels, and ROCKiY27632 on Spheroid Fusion

To form hHA hydrogels, 1% (w/v) HA from Sigma Aldrich solution wasreacted with a 5-fold molar excess amount of methacrylic anhydride (MA)from Sigma Aldrich for 15 h in the dark at 4° C. The final product wascollected by precipitating the solution in 5-fold volume of ethanoltwice, and was then purified by dialysis using a membrane (3.5 kDa Mwcut-off, Thermofisher) to remove unreacted reagents. The purified MA-HAwas filtered, lyophilized, and stored at −20° C. until further use [49].Next, ¹H-NMR spectroscopy (a Bruker 500M spectrometer at 200 MHz) wasused to analyze and the degree of methacrylation. For cultureexperiments, day 14 iEC spheroids were transferred to the wellscontaining day 14 iNPC spheroids, at the same time hMSCs were added intothe wells at different ratios (iNPC:iEC:MSC=4:2:0, 3:2:1 2:2:2, and1:2:3). The aggregates were cultured in medium with differentconcentrations of Geltrex (5%, or 10% v/v) or HA hydrogel (0.025 or 0.05wt %) or treated with ROCK inhibitor Y-27632 (20 or 40 μM) at day 0. Themorphology of hybrid spheroids were captured over 7 days by a phasecontrast microscope.

Images of spheroid fusion were captured over time by a phase contrastmicroscope. The captured images were converted to binary images usingImageJ software (rsb.info.nih.gov/ij) and analyzed with the “particleanalysis tool”. Through particle analysis, the squared aspect ratio ofcontact length between the two aggregates over maximum diameter (FIG.6A) was determined to indicate the aggregate fusion process. Theinter-sphere angle was measured as the intersecting angle between thetangent lines of two contacted spheroids to the touch point. For someexperiments, the number of branching points and the total tube length inthe area were evaluated.

Example 5: Analysis of Hybrid Spheroids

Biochemical Assays

First, an MTT assay was conducted. The spheroids of Examples 2 and 3were incubated with 5 mg/mL3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT,Sigma) solution at day 7 after three cell types were co-cultured unlessotherwise noted. The absorbance of the samples was measured at 500 nmusing a microplate reader (Biorad, Richmond, CA).

Next, a DNA assay was conducted. The DNA content of the hybrid spheroidsof Examples 2 and 3 was determined at day 7 after three cell types wereco-cultured unless otherwise noted. A DNA standard was prepared bydissolving salmon testes DNA in TEX (10 mM Tris, 1 mM EDTA, 0.1% TritonX-100 at pH 8) and a standard curve was constructed for each assay. Theaggregates were lysed with 0.1 mg/mL proteinase K (Fisher Scientific,Pittsburgh, PA) at 50° C. overnight. The lysates (100 μL) were mixedwith 100 μL of Picogreen (Molecular Probes) in a 96-well plate. Theplate was incubated for 5 min in the dark and then read on a fluorescentplate reader (FLX800, Bioinstrument Inc., Winooski, VT).

Next, an enzyme-linked immunosorbent assay (ELISA) assay was conducted.To quantify the growth factors secreted by different spheroids, culturesupernatants were collected at day 7 after three cell types wereco-cultured. Concentrations of FGF-2, PGE2, VEGF, and transforminggrowth factor (TGF)-β1 were measured by ELISA according to themanufacturers' instructions (R&D Systems, Minneapolis, MN for PGE2 andFGF-2; Life Technologies for TGF-β1 and VEGF). Briefly, the samples wereadded into 96-wps and incubated with primary/secondary antibody solutionconjugated with horseradish peroxidase for 2-3 hours. After washing,3,3′,5,5′-tetramethylbenzidine substrate solution was added, and themixture was incubated for 30 min. The absorbance units were measuredusing a microplate reader (Bio-Rad, Richmond, CA).

Immunocytochemistry of Hybrid Spheroids

The hybrid spheroids of Examples 2 and 3 were fixed with 4%paraformaldehyde (PFA) and permeabilized with 0.2-0.5% Triton X-100. Thesamples were then blocked for 30 min and incubated with various mouse orrabbit primary antibodies, listed in Table 1 below for four hours.

TABLE 1 Antibodies Primary Origin/ Cells Antibody Isotype Supplier/Cat#Dilution Neural Nestin Rabbit IgG Sigma, N5413 1:100 cells FOXG1 RabbitIgG ThermoFisher, 1:100 PA5-26794 TBR1 Rabbit IgG ABCAM, ab31940 1:200(layer VI) SATB2 Mouse IgG₁ ABCAM, ab51502 1:5   (layer IV) BRN2 GoatIgG Santa Cruz, sc-6029 1:200 (layer III) β-tubulin III Mouse IgG₁Millipore, MAB1637 1:200 MAP-2 Rabbit IgG ABCAM, ab32454 1:200 ISL1Rabbit IgG Millipore, AB4326 1:300 HOXB4 Rabbit IgG ABCAM, ab76093 1:200Glutamate Rabbit IgG Sigma, G6642  1:1000 GABA Rabbit IgG Sigma, A2052 1:1000 Synaptic Synapsin I Rabbit IgG Millipore, 574777 1:500 markersPSD95 Rabbit IgG Life Technologies, 1:200 51-6900 Astrocytes GFAP MouseIgG₁ Millipore, MAB360 1:400 Endothelial KDR Mouse IgG₁ Millipore,05-554 1:100 cells CD31 Goat Santa Cruz, sc-1506 1:200 polyclonal IgGVE- Goat Santa Cruz, sc-6458 1:200 cadherin polyclonal IgG ZO-1 MouseIgG₁ Life Technologies, 1:100 33-9100 Cell-cell E-cadherin Mouse IgG₁Millipore; MABT26 1:100 adhesion Collagen IV Rabbit IgG ABCAM, ab65861:200 Laminin Rabbit IgG ABCAM, ab11575 1:200 CSPG Mouse IgM LifeTechnologies, 1:100 MA1-83055 Hyaluronic Sheep IgG Life Technologies,1:50  acid PA1-85561 Pro- BrdU Mouse IgG₁ Life Technologies, 1:200liferation 03-3900 Secondary Alexa 488, — Life Technologies, 1:200 goatanti- A-21121 mouse IgG1 Alexa 488, — Life Technologies, 1:200 goatanti- A-11034 rabbit IgG Alexa 488, — Life Technologies, 1:200 goatanti- A-21042 mouse IgM Alexa 594, — Life Technologies, 1:400 donkeyanti- A-11016 sheep IgG Alexa 594, — Life Technologies, 1:400 goat anti-A-11012 rabbit IgG Alexa 594, — Life Technologies, 1:400 donkey A-11058anti-goat IgG

For surface markers, no permeabilization was performed. After washing,the cells were incubated with the corresponding secondary antibody:Alexa Fluor® 488 goat anti-Mouse IgG₁, Alexa Fluor® 488 or 594 goatanti-Rabbit IgG, or 594 donkey anti-goat IgG (Life Technologies) for onehour. The samples were counterstained with Hoechst 33342 and visualizedusing a fluorescent microscope (Olympus IX70, Melville, NY) or aconfocal microscope (Zeiss LSM 880).

Histology

For histology, various spheroids/organoids (day 21 or day 47, describedin Examples 2 and 3 above) were fixed in 10% formalin, dehydrated, andembedded in paraffin wax. Next, sections of 10 μm were cut and stainedwith Lerner-2 Hematoxylin (Lerner Laboratories, Pittsburgh, PA) andEosin-Y w/Phloxine (Richard-Allan Scientific, Kalamazoo, MI) [47]. Thesections were also stained with anti-CD31, β-tubulin III, zona occludens1 (ZO-1), HOXB4, TBR1, SATB2, BRN2, Collagen IV and laminin to showcellular distribution and cortical layer formation. Images were capturedwith an Olympus IX70 microscope with MagnaFire SP 2.1B software or aconfocal microscope (Zeiss LSM 880).

Hybrid spheroids were re-plated to investigate their cellularcomposition (FIGS. 21A-21D).

The control images for histology images in FIGS. 21A-21D were producedwith BRN2/Hoechst and MAP2/Hoechst for iNPC spheroids and CD31/Hoechst,VE-cadherin/Hoechst, and ZO1/Hoechst for iEC spheroids (not shown).Enlarged images for neural marker β-tubulin III/vascular marker CD31expression in hybrid spheroids of FIG. 22A was produced (not shown).

The expression of vascular markers, CD31 and VE-cadherin, was observedfor all the groups. The fused spheroids also expressed ZO-1, the tightjunction protein of brain microvascular cells (not shown). Co-stainingof CD31 and Nestin showed that CD31⁺ cells interacted with the Nestin⁺neural cells (not shown). The expression of deep cortical layer VImarker TBR1 (and a little BRN2 expression) indicated the corticalidentity of the hybrid spheroids, although hindbrain marker HOXB4 wasalso expressed at this stage.

Specifically, the neural and vascular marker expression of hybridspheroids was depicted (not shown). Day 21 hybrid spheroids werereplated for three days and immunocytochemistry was performed.Fluorescent images of vascular markers, including CD31/Hoechst forendothelial cells, VE-cadherin/Hoechst for later stage of endothelialcells, and ZO1/Hoechst, the tight junction protein expressed by brainmicrovascular endothelial cells were produced (not shown). Fluorescentimages of neural markers, including Nestin for neural progenitors, HOXB4(a hindbrain marker), TBR1 (forebrain deep cortical layer VI) and BRN2(forebrain cortical superficial layer II-IV) were produced (not shown).Fluorescent images of MAP2 (more mature neurons), GFAP (astrocyteprogenitors) and E-cadherin (cell-cell interactions) were produced (notshown). Extensive MAP2 signals showed the mature neuron population,while the expression of GFAP indicated the existence of glialprogenitors. Cells from tri-cultured spheroids had more E-cadherinexpression (heterogeneous signal intensity), while Bi-(4:2) group hadhomogenous expression.

Histological sections were evaluated to assess the in situ distributionand localization of iECs and neural cells within the hybrid spheroids(not shown). Numerous CD31⁺ vascular cells interacted with β-tubulinIII⁺ neurons were observed throughout the spheroids (not shown). Inaddition, the distribution of CD31 was more homogenous for Tri-(2:2:2)and Tri-(1:2:3) groups. More ZO-1 expression was observed fortri-cultured spheroids. Confocal images of intact spheroids of FOXG1 andCD31 showed the FOXG1⁺ layers and the lumens of CD31⁺ cells (not shown).The expression of TBR1 inside the fused spheroids was observed, but theexpression of BRN2 was minimal at this early stage (not shown).

Specifically, FIG. 15 depicts morphology and histology characterizationsfor early (day 21) and late (day 47) stage hybrid spheroids. Images ofhistology thin sections of day 21 spheroids were produced using corticallayer marker TBR1/BRN2, HOXB4 and Hoechst (not shown). Phase contrastimages of hybrid spheroid morphology at day 40 (FIG. 15 ). Scale bar:400 μm. Collagen IV, laminin, chondroitin sulfate proteoglycans (CSPG),and hyaluronic acid (HA) expression for hybrid spheroid histologicalsections at day 47 was observed (not shown).

In addition, one side of spheroids expressed HOXB4. The quantificationof 3-tubulin III showed the higher level for tri-cultured spheroids(i.e., 47.2±6.7%, 50.2±3.2%, 63.4±6.8% for Tri-(3:2:1), Tri-(2:2:2),Tri-(1:2:3) respectively) than Bi-(4:2) group (18.9±12.8%) (FIGS. 21A,21C).

Specifically, FIGS. 21A-21D depict histology images of early and latestage hybrid spheroids and quantification by flow cytometry. Images ofhistology thin sections of spheroids (total day 21) showed theexpression of β-tubulin III and vascular marker CD31, ZO1 (not shown).Confocal images of histology thin sections of spheroids (total day 47)for β-tubulin III and vascular marker CD31 were taken (not shown).Confocal images of day 21 spheroids for FOXG1 and CD31 were taken (notshown). Flow cytometry histograms for day 21 iEC spheroids, iNPCspheroids, or tri-cultured spheroids are depicted for β-tubulin III(FIG. 21A) and CD31 (FIG. 21B). Flow cytometry quantification ofβ-tubulin III (β-tub III, n=3) (FIG. 21C). Flow cytometry quantificationof CD31 (n=3) (FIG. 21D). *indicates p<0.05 for the different testconditions. Images of histology thin sections of late stage spheroids(total day 47) showed the expression of cortical layer markers:TBR1/SATB2, BRN2/SATB2 (not shown).

Similarly, higher CD31 expression was observed for the tri-culturedspheroids (i.e., 38.9±5.7%, 55.8±15.8%, 61.1±1.2% for Tri-(3:2:1),Tri-(2:2:2), Tri-(1:2:3) respectively) than Bi-(4:2) group (28.6±1.8%)(FIGS. 21B, 21D). Late stage histology of day 47 forebrain organoidsshowed the development of superficial cortical layer II-IV indicated bySATB2 and BRN2 expression (FIG. 15 ). For Bi-(4:2), SATB2 layer wasunder the TBR1 layer, while the two layers mixed together forTri-(3:2:1) and Tri-(2:2:2). For Tri-(1:2:3), the TBR1 layer movedtoward the center of organoids while the SATB2 layer moved to thesurface, according to “inside-out” cortical layer development [32]. Basemembrane proteins Collagen IV and laminin as well as brain matrixchondroitin sulfate proteoglycans and hyaluronic acid were detected inday 47 hybrid organoids (not shown). These results indicate thattri-culture promotes neural and vascular differentiation, and the betterratio is Tri-(1:2:3) group.

Flow Cytometry

To quantify the levels of various markers, the cells were harvested bytrypsinization and analyzed by flow cytometry [50]. Briefly, 1×10⁶ cellsper sample were fixed with 4% PFA and washed with staining buffer (2%FBS in PBS). The cells were permeabilized with 100% cold methanol,blocked, and then incubated with primary antibodies against β-tubulinIII, KDR, CD31, and VE-cadherin, followed by the corresponding secondaryantibody Alexa Fluor 488 goat anti-Mouse IgG₁ (for β-tubulin III, KDR),or Alexa Fluor 594 donkey anti-goat IgG (for CD31, VE-Cadherin [51]).For surface markers, no permeabilization was performed. The cells wereacquired with BD FACSCanto™ II flow cytometer (Becton Dickinson) andanalyzed against isotype controls using FlowJo software.

Reverse Transcription Polymerase Chain Reaction (RT-PCR) Analysis

Total RNA was isolated using the RNeasy Mini Kit (Qiagen, Valencia, CA)according to the manufacturer's protocol followed by the treatment ofDNA-Free RNA Kit (Zymo, Irvine, CA) [52]. Reverse transcription wascarried out using 2 μg of total RNA, anchored oligo-dT primers (Operon,Huntsville, AL), and Superscript III (Invitrogen, Carlsbad, CA)(according to the protocol of the manufacturer). Primers specific fortarget genes were designed using the software Oligo Explorer 1.2(Genelink, Hawthorne, NY), and are shown in Table 2 below:

TABLE 2 Primer Sequences for Target Genes Gene Forward primer 5′ to 3′Reverse primer 5′ to 3′ TBR1 CCCCCTCGTCTTTCTCTTACC TAATGTGGAGGCCGAGACTTGHOXB4 AATTCCTTCTCCAGCTCCAAGA CCTGGATGCGCAAAGTTCA Nkx2.1GAGTCCAGAGCCATGTCAGC GCATAAAACAGCTTTGGGGTGT MMP2 CATCGCTCAGATCCGTGGTGGCATCAATCTTTTCCGGGAGC MMP3 CCATCTCTTCCTTCAGGCGT ATGCCTCTTGGGTATCCAGCNotch-1 CACTGCTGCCCTCCCCGTTC TTCAGGTGCCCGATGCCCAG GLUT-1AGCAACTGTGTGGTCCCTACG AAGGTCCGGCCTTTAGTCTCA BCRP CAGGTGTGCGTCAGAATCATCTCCAGGAGTGGTCAGATTCCTT PGP ACCACTCTCCCACCTCCCTTA TTTAGCTGGGCTGCGTTTACAß-actin GTACTCCGTGTGGATCGGCG AAGCATTTGCGGTGGACGATGG

The gene 3-actin was used as an endogenous control for normalization ofexpression levels. Real-time RT-PCR reactions were performed on anABI7500 instrument (Applied Biosystems, Foster City, CA), using SYBR1Green PCR Master Mix (Applied Biosystems). The amplification reactionswere performed as follows: 2 min at 50° C., 10 min at 95° C., and 40cycles of 95° C. for 15 sec and 55° C. for 30 sec, and 68° C. for 30sec. Fold variation in gene expression was quantified by means of thecomparative Ct method: 2^(−(C) ^(t treatment) ^(-C) ^(t control) ⁾,which is based on the comparison of expression of the target gene(normalized to the endogenous control β-actin) between the hybridspheroids and the spheroids of NPC only.

Gene expression of brain regional markers TBR1, HOXB4, and Nkx2.1 weredetermined for day 21 hybrid spheroids (FIGS. 22A-22I and 17A-17C).Specifically, FIGS. 17A-17C depict RT-PCR analysis for iEC and hMSCcontrols compared to iNPC group. Brain regional marker genes; TBR1 andNkx2.1 were the highest for the iNPC group as expected (FIG. 17A). ForHOXB4, it may not be a specific hindbrain marker. Matrix remodeling andcell-cell communication genes; MMP2 and MMP3 were the highest for theMSC group as expected, then iEC group, and iNPC group (FIG. 17B).Blood-brain barrier-related genes (FIG. 17C). GLUT-1 and PGP were thehighest for the iNPC group, lowest for the MSC group. The iECsdifferentiation protocol used in this study generate the comment ECs,not the brain microvascular ECs. *indicates p<0.05.

The expression level of cortical deep layer VI marker TBR1 increasedwith the abundance of hMSCs in the hybrid spheroids, i.e., 1.74±0.11,1.63±0.17, 3.39±0.22, 4.88±0.38 fold for Bi-(4:2), Tri-(3:2:1),Tri-(2:2:2), Tri-(1:2:3) respectively (FIG. 22A).

Specifically, FIGS. 22A-22I depict the results of RT-PCR analysis ofgene expression of hybrid spheroids. mRNAs were isolated from day 21hybrid spheroids for RT-PCR. Brain regional marker genes (contributionfrom iNPCs): TBR1 (FIG. 22A), Nkx2.1 (FIG. 22B); HOXB4 (FIG. 22C).Matrix remodeling and cell-cell communication genes (contribution fromMSCs): MMP2 (FIG. 22D); MMP3 (FIG. 22E); Notch-1 (FIG. 22F). Blood-brainbarrier-related genes (contributions from iECs): GLUT-1 (FIG. 22G); BCRP(FIG. 22H); PGP (FIG. 22I). *indicates p<0.05 for the test conditionscompared with the iNPC only control. # indicates p<0.05 among the testconditions.

Similarly, significantly higher expression of Nkx2.1, a ventral regionalmarker, was observed for hybrid spheroids, i.e., 14.1±1.3, 6.7±1.4,11.1±4.8, 50.5±1.8 fold for Bi-(4:2), Tri-(3:2:1), Tri-(2:2:2),Tri-(1:2:3) respectively than iNPC-only spheroids (FIG. 22B). However,HOXB4 was similarly expressed for all groups (FIG. 22C).

Matrix metalloproteinases (MMPs) play a critical role in neural cellproliferation, migration and differentiation [60-62]. Significantlyhigher expression of MMP2 was observed for hybrid spheroids than theiNPC only spheroids, i.e., 1.28±0.10, 2.02±0.13, 1.96±0.43, 3.90±0.19fold for Bi-(4:2), Tri-(3:2:1), Tri-(2:2:2), Tri-(1:2:3) respectively(FIG. 22D). For MMP3, higher expression was observed for hybridspheroids than iNPC only spheroids except for Tri-(3:2:1), i.e.,2.60±0.19, 1.36±0.85, 4.06±0.76, 8.05±0.09 fold for Bi-(4:2),Tri-(3:2:1), Tri-(2:2:2), Tri-(1:2:3) respectively (FIG. 22E). Notchsignaling is known to enhance cell-cell communications and involved inthe BBB formation. Upregulation of Notch-1 expression was observed inthe hybrid spheroids, i.e., 3.1±0 0.48, 2.17±0.51, 4.38±0.91, 8.72±0.65fold for Bi-(4:2), Tri-(3:2:1), Tri-(2:2:2), Tri-(1:2:3) respectively(FIG. 22F). These results suggest that incorporation of MSCs upregulatedMMP2, MMP3, and Notch-1 expression.

The expression of glucose transporter 1 (GLUT-1) and effluxtransporters, BCRP and PGP, was determined to demonstrate whether theiECs possess BBB properties. For GLUT-1, higher expression was observedfor all tri-cultured groups compared to iNPC only spheroids and Bi-(4:2)group, i.e., 0.58±0.01, 2.10±0.004, 3.23±0.07, 2.72±0.02 fold forBi-(4:2), Tri-(3:2:1), Tri-(2:2:2), Tri-(1:2:3) respectively (FIG. 22G).BCRP was dependent on the abundance of the hMSCs, and the Tri-(1:2:3)group showed the highest BCRP gene expression, i.e., 0.80±0.03,0.87±0.08, 1.26±0.10, 1.62±0.04 fold for Bi-(4:2), Tri-(3:2:1),Tri-(2:2:2), Tri-(1:2:3) respectively (FIG. 22H). For PGP, higherexpression was observed for Tri-(2:2:2) and Tri-(1:2:3) groups comparedto Tri-(3:2:1) group, i.e., 1.00±0.09, 0.83±0.06, 0.98±0.08, 1.06±0.00fold for Bi-(4:2), Tri-(3:2:1), Tri-(2:2:2), Tri-(1:2:3) respectively(FIG. 22I). These observations indicate that co-culturing iECs withiNPCs and hMSCs increased the expression of GLUT1 and BCRP.

DNA content of hybrid spheroids was measured on merged day 1. Bi-(4:2)group showed the highest DNA content, while the DNA content in the otherthree groups was comparable. Specifically, FIGS. 11A-11D depict themetabolic activity and DNA content of hybrid spheroids in suspension:DNA content of hybrid neural spheroids after 1 or 7 days of tri-culture(total day 15, 21) (FIG. 11A); DNA content of hybrid spheroids with thetreatment of 5% Geltrex after 7 days (total day 21) of tri-culturecompared to the control (no treatment) (FIG. 11B); DNA content of hybridspheroids with the treatment of 0.05 wt % hyaluronic acid (HA) after 7days (total day 21) of tri-culture compared to control group (notreatment) (FIG. 11C); and MTT activity of hybrid spheroids with thetreatment of 20 μM ROCKi Y27632 after 7 days (total day 21) oftri-culture compared to control group (no treatment) (FIG. 11D).*indicates p<0.05 for the different test conditions.

After 7 days of culture, all four groups showed cell proliferation withincreased DNA content (FIG. 11A). Increased DNA content was observed forTri-(2:2:2) and Tri-(1:2:3) spheroids with the treatment of 5% Geltrex(FIG. 11B). The treatment of 0.05 wt % HA showed no significantdifference in DNA content except for Bi-(4:2) spheroids, while 0.025 wt% HA treatment showed no significant difference in DNA content for allthe groups (FIG. 11C and FIG. 12 ). Higher MTT activity was observed forthe Bi-(4:2) spheroids compared to the other three groups, and thetreatment of ROCKi increased MTT activity (FIG. 11D).5-Bromo-2′-deoxyuridine (BrdU) assay showed that the cells in S-phase ofcell cycle were not homogeneously happening in the spheroids, but tendedto be localized to the interface of the spheroids or the spheroidsurface (FIG. 12 ). Specifically, FIG. 12 shows the DNA content ofhybrid spheroids with the treatment of 0.025 wt % hyaluronic acid (HA)after 7 days (total day 21) of tri-culture compared to control group (notreatment). Confocal images of 5-Bromo-2′-deoxyuridine (BrDU), showingcells in S-phase of cell cycle were produced (not shown). For5-Bromo-2′-deoxyuridine (BrdU) assay, the cells were incubated in mediumcontaining 10 μM BrdU (Sigma) for four hours. The cells were then fixedwith 70% cold ethanol, followed by a denaturation step using 2N HCl/0.5%Triton X-100 for 30 min in the dark. The samples were reduced with 1mg/mL sodium borohydride for 5 min and incubated with mouse anti-BrdU(1:100, Life Technologies) in blocking buffer (0.5% Tween 20/1% bovineserum albumin in PBS), followed by Alexa Fluor® 488 goat anti-Mouse IgG1(Molecular Probes). The cells were counterstained with Hoechst 33342 andanalyzed by a confocal microscope (Zeiss LSM 880).

Without intending to be bound by any particular theory, it is believedthat the trophic factors secreted by MSCs (e.g., FGF-2 and VEGF-A) canenhance the angiogenesis, neurogenesis, and axonal growth during neuraltissue regeneration [56, 57]. MSCs also secret anti-inflammatoryfactors, such as TGF-β1 and PEG2, to regulate the immune response [58,59]. The secretion levels of FGF-2, VEGF-A, PGE2, and TGF-β1 from thehybrid spheroids of Examples 2 and 3 was characterized (FIGS. 13A-13D).

Specifically, FIGS. 13A-13D depict cytokine secretion by hybridspheroids during neural differentiation from culture supernatantscollected and measured by enzyme-linked immunosorbent assay (ELISA) fordifferent spheroids at day 21. Concentrations of FGF2 (FIG. 13A), VEGF-A(FIG. 13B), PGE2 (FIG. 13C), and TGF-β1 (FIG. 13D). iNPC only indicatesday 21 iNPC spheroids; MSC only indicates day 7 MSC spheroids.*indicates p<0.05 for the test conditions compared with the iNPC onlycontrol. # indicates p<0.05 among the test conditions. $ indicatesp<0.05 for the test conditions compared with the MSC only control.

The highest secretion levels were observed for the hMSC-only group. ForFGF-2, higher secretion was observed for Tri-(2:2:2) spheroids comparedto iNPC-only spheroids (FIG. 13A). The VEGF-A concentration increasedwith the relative ratio of hMSCs in the hybrid spheroids (FIG. 13B andFIG. 14 ). Similarly, the incorporation of hMSCs in the neural spheroidsupregulated PGE2 secretion compared to iNPC-only and Bi-(4:2) groups(FIG. 13C). Tri-(3:2:1) and Tri-(1:2:3) had higher TGF-β1 concentrationthan the iNPC-only group (FIG. 13D). Without intending to be bound byany particular theory, it is believed that these results indicate thattri-cultures do not promote cytokine secretion, but that cytokinessecretion from MSCs is maintained in hybrid spheroid culture in anamount depending on the MSC ratio used.

Effect of AMD3100

The Day 14 spheroids of Example 2 were labeled with CellTracker Red. ThehMSCs were labeled with CellTracker Green. The hybrid (iNPC-iEC-MSC)spheroids were cultured in neural differentiation media (control) ormedia containing the CXCR4 inhibitor AMD3100 (100 nM, Sigma) foradditional 10 days [53]. The fusion kinetics and cell localization werecaptured over time. The cell viability of day 10 hybrid spheroids wasdetermined by MTT activity assay.

The influence of Geltrex, HA, and Y-27632 on the neurite morphology wasexamined (FIGS. 18A-18B). Specifically, FIGS. 18A-18B depictinvestigation of neurogenesis of hybrid iNPC-MSC spheroids on 2D surfacewith iEC layer. Day 21 hybrid iNPC-MSC spheroids were replated overper-cultured iECs for three days and immunocytochemistry was performedfor neural marker β-tubulin III (not shown). Phase contrast images ofaxon outgrowth of day 21 hybrid iNPC-MSC spheroids replated overper-coated endothelial cells (not shown). Average neurite lengths wereanalyzed and shown in FIGS. 18A-18B, respectively. *indicates p<0.05 forthe different test conditions.

Effects of ROCKi Y27632, Geltrex, and HA on neurogenesis of hybridspheroids were depicted (not shown). Day 21 hybrid spheroids embedded inGeltrex (5%) or treated with ROCKi Y27632 (10 μM) were replated on 1%Geltrex-coated surface for three days. Neurite and axonal extensionindicated by β-tubulin III expression was shown. Phase contrast imagesof axon outgrowth of day 21 spheroids were produced (not shown).Fluorescent images for day 39 spheroids: neuron marker β-tubulin III,motor neuron progenitor marker Islet-1, GABAergic neuron marker (GABA),glutamatergic neuron marker (Glutamate), cortical neuron marker (TBR1),pre-synaptic marker synapsin I and post-synaptic marker PSD95 wereproduced (not shown).

The neurite outgrowth was enhanced by Y27632, showing more β-tubulinIII⁺ axons and denser axons. Geltrex promoted the neurite outgrowth, butthe packing density of axon was not high. Prolonged corticaldifferentiation was performed for day 39 hybrid spheroids treated withGeltrex or 0.025 wt % HA (not shown). The hybrid spheroids containedGABAergic neurons and Glutamatergic neurons, and expressed pre-synapticmarker synapsin I and post-synaptic marker PSD95.

It has been reported that cell migration in cerebral organoids dependson CXCR4 (a cell homing receptor) activity [12]. To understand thespheroid fusion mechanism, the effects of CXCR4 antagonist, AMD3100, onspheroid fusion was investigated (FIGS. 23A-23D). Specifically, FIGS.23A-23D depict the effects of AMD3100 treatment on the aggregationkinetics of hybrid spheroids. Overlay of phase contrast images (iNPCs)with fluorescent images (hMSCs labeled with CellTracker Green, iECslabeled with CellTracker Red) of different hybrid spheroids were eitheruntreated (control) (FIG. 23A) or treated with CXCR4 inhibitor (AMD3100)(FIG. 23B). Scale bar: 400 μm. Analysis of aspect ratios, i.e., area ofMSCs in fused spheroids over total area of fused spheroid, for groupseither untreated (control) (FIG. 23C) or treated with CXCR4 inhibitor(AMD3100) (FIG. 23D). *indicates p<0.05 for the different testconditions.

The MTT activity indicated that AMD3100 treatment had little influenceon cell proliferation (FIG. 19 ). FIG. 19 depicts the effects of AMD3100treatment on MTT activity of hybrid spheroids. The day 24 (after threecell types were put together) hybrid spheroids were measured for MTTactivity assay after AMD3100 treatment. *indicates p<0.05 for thedifferent test conditions.

iEC spheroids (with CellTracker Red) gradually fused with the iNPCspheroids. Small hMSC (with CellTracker Green) areas sparsely spreadover the fused spheroids for Tri-(3:2:1) group. For Tri-(2:2:2) andTri-(1:2:3) groups, one large area of hMSCs occupied the interface ofiEC and iNPC spheroids (FIGS. 23A, 23C). For AMD3100 treatment, the areaoccupied by hMSCs was smaller than the control groups (FIGS. 23B, 23D).Analysis of the relative ratio of area occupied by hMSCs to the totalarea of fused spheroids showed that the aspect ratio of hMSCs decreasedfrom day 4 to day 8 with AMD3100 treatment (FIG. 23D). Without intendingto be bound by any particular theory, it is believed that these dataindicate that AMD3100 may inhibit hMSC invasion during spheroid fusion.

Whole-Patch Clamping for Electrophysiology

A whole-cell patch clamp was used to record from the spheroids ofExamples 2 and 3 cultured on glass covered slips. Cover slips werewashed three times with extracellular recording solution containing (inmM) 136 NaCl, 4 KCl, 2 MgCl, 10 HEPES, and 1 EGTA (312 mOsm, pH 7.39)and were incubated in this solution at room temperature duringrecording. Glass electrodes (resistance 1-5 MQ) were filled withintracellular solution containing 130 mM KCl, 10 mM HEPES, and 5 mM EGTA(292 mOsm, pH 7.20). Cells were visualized under phase contrast with aNikon Eclipse Ti-U inverted microscope and attached DS-Qil monochromedigital camera. Recordings were made with an Axopatch 200B amplifier(Molecular Devices) and digitized with a Digidata 1440A system(Molecular Devices). Ionic currents were recorded under a voltage clampprotocol (−60 mV to 135 mV in 15 mV steps, 250 ms in duration). Actionpotentials were recorded under a current clamp protocol (−100 pA to 200pA in 20 pA steps, 800 ms in duration). Spontaneous post-synapticcurrents were recorded under continuous voltage clamp at −80 mV for 2min. Signals were filtered at 1 kHz and sampled at 10 kHz. Data wascollected and analyzed using pCLAMP 10 software (Molecular Devices).

The electrophysiological properties of the outgrowth cells of thederived spheroids/organoids were examined via patch clamping. As cellswithin the dense core of the spheroid cannot be visualized by phasecontrast microscopy while in the recording chamber, outgrowth cellstoward the boundary of the spheroid were chosen for these experiments.This allowed us to simultaneously evaluate the electrical activity andcellular morphology of the recorded cells for neuron-like properties.Recorded cells displayed fast inward currents and long-lasting outwardcurrents during voltage-clamp recording, suggesting the presence offunctional voltage-gated Na⁺ and K⁺ channels, respectively (FIGS.16A-16D).

Specifically, FIGS. 16A-16D depict electrophysiological properties ofhybrid spheroids (day 42). Results were from Bi-(4:2) group.Representative voltage clamp traces showing fast inward Na+ currents andlong-lasting outward K+ currents evoked by depolarizing voltage steps(FIG. 16A). Step size=15 mV. Representative current clamp traces showingrebound action potentials in response to hyperpolarizing currentinjections (FIG. 16B). Step size=20 pA. Representative trace ofcontinuous voltage clamp recording showing spontaneous postsynapticcurrents (FIG. 16C). Representative phase-contrast images of a recordedneuron outgrowth of the spheroids (FIG. 16D). Scale bar: 20 μm.

In addition, a subpopulation of the cells fired rebound actionpotentials in response to hyperpolarizing current injection duringcurrent clamp recording. Spontaneous postsynaptic currents were observedin the absence of stimulation during continuous voltage clamp recording.Cellular morphology was stereotypically neuron-like, with small cellbodies and extensive long and thin projections. Together, these resultssuggest that the hybrid spheroids have the functional and morphologicalproperties of neurons including synaptic activity.

While the disclosure has been described with reference to a number ofembodiments, it will be understood by those skilled in the art that theinvention is not limited to such disclosed embodiments. Rather, theinvention can be modified to incorporate any number of variations,alterations, substitutions, or equivalent arrangements not describedherein, but which are commensurate with the spirit and scope of theinvention. Additionally, while various embodiments of the invention havebeen described, it is to be understood that aspects of the invention mayinclude only some of the described embodiments. Accordingly, theinvention is not to be seen as limited by the foregoing description, butis only limited by the scope of the appended claims.

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That which is claimed is:
 1. A hybrid neurovascular spheroid comprisingcortical cells, endothelial cells, and mesenchymal stem cells.
 2. Thehybrid neurovascular spheroid of claim 1, wherein the hybridneurovascular spheroid displays upregulated secretion levels of one ormore cytokines VEGF-A, PGE2, and TBF-β1 relative to a comparative hybridneurovascular spheroid prepared by directly mixing cultures of corticalcells, endothelial cells, and mesenchymal stem cells.
 3. The hybridneurovascular spheroid of claim 1, wherein the hybrid neurovascularspheroid displays increased expression of one or more of TBR1, Nkx2.1,matrix remodeling, MMP2, MMP3, Notch-1, GLUT-1, CD31, or ZO-1 genesrelative to a comparative hybrid neurovascular spheroid prepared bydirectly mixing cultures of cortical cells, endothelial cells, andmesenchymal stem cells.
 4. The hybrid neurovascular spheroid of claim 1,wherein the cortical cells are human iPSC-derived cortical neuralprogenitor cells.
 5. The hybrid neurovascular spheroid of claim 1,wherein the endothelial cells are human endothelial cells.
 6. The hybridneurovascular spheroid of claim 1, wherein the mesenchymal stem cellsare human mensenchymal stem cells (hMSC).
 7. The hybrid neurovascularspheroid of claim 1, wherein the cortical cells are human inducedpluripotent stem cells (iPSC)-derived cortical neural progenitor cells,and wherein the endothelial cells are human endothelial cells.
 8. Thehybrid neurovascular spheroid of claim 1, wherein the cortical cells arehuman iPSC-derived cortical neural progenitor cells, and wherein themesenchymal stem cells are human mensenchymal stem cells (hMSC).
 9. Thehybrid neurovascular spheroid of claim 1, wherein the cortical cells arehuman iPSC-derived cortical neural progenitor cells, wherein theendothelial cells are human endothelial cells, and wherein themesenchymal stem cells are human mensenchymal stem cells (hMSC).
 10. Thehybrid neurovascular spheroid of claim 1, wherein a ratio based oninitial seeded cell numbers of the cortical cells to the endothelialcells to the mesenchymal stem cells is from about 4:2:0 to about 1:2:3.11. The hybrid neurovascular spheroid of claim 1, wherein one or more ofthe cortical cells, endothelial cells, and mesenchymal stem cells arepropagated in a hydrogel comprising basement membrane, hyaluronic acid,or any combination thereof.
 12. The hybrid neurovascular spheroid ofclaim 1, wherein the hybrid neurovascular spheroid is anantagonist-treated hybrid neurovascular spheroid.
 13. A hybridneurovascular spheroid comprising cortical cells, endothelial cells, andmesenchymal stem cells, wherein the hybrid neurovascular spheroiddisplays upregulated secretion levels of one or more cytokines VEGF-A,PGE2, and TBF-β1 relative to a comparative hybrid neurovascular spheroidprepared by directly mixing cultures of cortical cells, endothelialcells, and mesenchymal stem cells.